Cinnamyl alcohol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2020 to June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamyl alcohol

EC Number:

203-212-3

EC Name:

Cinnamyl alcohol

Cas Number:

104-54-1

Molecular formula:

C9H10O

IUPAC Name:

3-phenylpropan-1-ol

Test material form:

solid

Remarks:

White to yellow

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

In-house bred.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were 9 weeks of age at receipt. Females were nulliparous and non-pregnant.
Body weight at receipt :
Males: 250.25 to 297.51 g
Females: 200.85 to 249.90 g
Animals were housed under standard laboratory conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.6 to 23.2oC and relative humidity 46 to 65%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, two animals of same sex were housed.
- Pre-mating – In each cage, two animals of the same sex and group were housed.
- Cohabitation Period (mating) – In each cage, two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming presence of sperm in the vaginal smear and/or vaginal plugs (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for mated females from gestation day 20 onwards.

Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co.KG) was available ad libitum to the animals throughout the experimental period. The contaminant analysis test report of the feed is included as Annexure 2. A sample of feed from the batch used in the study was retained until the finalization of study report and was discarded on the day of finalization of study report.

Water was available ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item/vehicle was administered by oral (gavage) route using stainless steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 5 mL/kg with the concentration of 17.5, 35 and 70 mg/mL for low, mid and high dose groups, respectively. Vehicle was administered to the control group at an equivolume of 5 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation

Details on mating procedure:

The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. Each femalerat was housed with its respective male rat until pregnancy occured by evidence of sperm in the vaginal smear or until two weeks. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear. Four females one each from groups G1, G2 and two from group G4 were not mated during the 14 days cohabitation period. These females were paired with a proven male on the day 14 of cohabitation till day 17 for animals from groups G1, G2 and till day 15 for the animal from group G4. The females confirmed with mating but not littered were sacrificed on day 25 after gestation day ‘0’.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1588 and the results are presented in the report. Sampling and analysis of the formulations were performed during week 1 and week 5 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The exact volumes of test item formulation samples are included in the study report. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation were analyzed. The second aliquot was stored for backup purpose at established stability conditions. The second set of samples were discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Duration of treatment / exposure:

The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). Thefemales were treated for two weeks pre-mating period, during mating and pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on PND 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

G4

Doses / concentrations 2

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

G3

Doses / concentrations 3

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Remarks:

G2

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (G1)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose levels were selected based largely on the results of an OECD 414 with cinnamaldehyde (CAS 104-55-2) (Sustainability Support Services Europe AB, Study No 19_49_143). In this OECD 414 study, unexpected mortality was observed at 500 mg/kg bw/day (1 dam on GD 9) and at 1000 mg/kg bw/day (multiple dams from GD 6 and onwards). Dose levels in the study after inclusion of an additional dose group were 0, 125, 250, 500 and 1000 mg/kg bw/day. NOAEL for general maternal toxicity was established at 250 mg/kg bw/day due to significant/severe toxicity at 500 mg/kg bw/day, which included: one death, clinical signs of toxicity (at least one of the following symptoms were observed in all animals treated at 500 mg/kg bw/day: hypothermia, lethargy, prostration, and excessive salivation), biologically significant changes in body weight on GD 20, and gross and microscopic lesions in stomach and lung. Based on these data and considering the longer exposure duration in an OCD 421 study compared with an OECD 414 study, a top dose of 350 mg/kg bw/day was selected in present study. This dose level was expected to produce clinical signs of toxicity, biologically/statistically significant decreases in body weight, and pathological lesions, but no mortality or suffering. Both cinnamyl alcohol (CAS 104-54-1) and cinnamaldehyde (CAS 104-55-2) are aromatic monocyclic compounds. The basic structure of these two substances is the monocyclic benzene moiety with a three-carbon unsaturated (i.e., at the 2,3- position) chain. Cinnamyl alcohol has a hydroxy group at the end of the chain, while cinnamaldehyde has an aldehyde group at the same position. The metabolic pathway of cinnamyl alcohol and cinnamaldehyde is also well-described (Bicker, et al., Food and Chemical Toxicology, 43 (2005) 799–836 and JECFA, 2000. WHO Food Additives Series: 46). Cinnamyl alcohol and cinnamaldehyde are both rapidly absorbed from the gut, metabolized and excreted primarily in the urine and to a minor extent, in faeces. Cinnamyl alcohol is rapidly converted to aldehyde via alcohol dehydrogenase to cinnamaldehyde, which would be converted to cinnamic acid in turn. Thus, cinnamic acid is the major intermediate metabolite for both chemicals. On this basis, the toxicological data on cinnamaldehyde in the mentioned OECD 414 study was considered appropriate and sufficient for dose level selection in the presented OECD 421 study with cinnamyl alcohol.

- The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -12.73 to 14.55%; Females: -14.11 to 8.69%) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment by randomization based on the body weights. Body weight of the animals were analyzed statistically for mean body weight to rule out statistically significant differences between groups within each sex prior to dosing.

Positive control:

Not included.

Examinations

Parental animals: Observations and examinations:

Mortality, clinical signs of toxicity, body weight, food intake, gestation length, and pre-coital interval.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).

Litter observations:

Number of pups per litter, sex ratio, live birth index, pup survival, pup weight, anogenital distance, male pup nipple/areolae retention.

Postmortem examinations (parental animals):

Uteri observations, hormone levels, organ weight, gross pathology and histopathology as per OECD 421 (2016).

Postmortem examinations (offspring):

Gross pathology

Statistics:

STATISTICAL ANALYSIS
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.

Note: Data of non-pregnant females were presented in the individual animal data but excluded for mean calculations and statistical analysis. The data of females mated but not littered and lactation data of females with total litter loss were presented in individual animal data and considered for mean calculations and statistical analysis where applicable.
The statistical analysis was followed but not limited to the parameters as mentioned below table.

Reproductive indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, and parturition index.

Offspring viability indices:

Live birth index and pup survival indexes on PND 4, 7, and 13.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity in the study.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant changes in body weight or in body weight gain with respect to day 1 of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes were observed except for statistically significant decreases in food intake between GD 7-14 (mean 20.52 g/rat/day) and between 14 to 20 (mean, 23.25 g/rat/day) in G4 dams compared to G1 dams (mean, 23.07 g/rat/day between GD 7-14 and mean, 26.34 g/rat/day between GD 14-20). These effects were considered to be non-adverse since no significant changes in body weight or body weight gain were observed during gestation or lactation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant changes in serum thyroxine (T4) hormone levels were observed (examined in G1, G2, G3 and G4 male rats).

Endocrine findings:

no effects observed

Description (incidence and severity):

No significant changes in serum thyroxine (T4) hormone levels were observed (examined in G1, G2, G3 and G4 male rats).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic findings were restricted to minimal degeneration of seminiferous tubules in testes observed in two males treated at 350 mg/kg. This finding was considered incidental as the changes were of minimal severity, distributed in focal areas, and unilateral in nature, and as such changes are commonly observed in laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of females in any of the tested dose groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in any of the tested dose groups when compared with vehicle control group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Mating Index (%): A total of 11 (out of 12), 11 (out of 12), 12 (out of 12) and
10 (out of 12) males were confirmed with mating with a mating index of 91.7%,
91.7%, 100.0% and 83.3% from group G1, G2, G3 and G4, respectively. There were
no statistically significant differences noted in male fertiltiy index at any of the tested dose groups when compared with vehicle control group.

Male Fertility Index (%): A total of 11 (out of 12), 11 (out of 12), 12 (out of 12) and
10 (out of 12) males were confirmed with impregnating a female with a fertiltiy index of 91.7%, 91.7%, 100.0% and 83.3% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted in male fertiltiy index at any of the tested dose groups when compared with vehicle control group.

Female Mating Index (%): All the 12 females from each tested dose group (G2, G3 and G4) and vehicle control group (G1) were confirmed with mating with a mating index of 100% for each group.

Female Fertility Index (%): A total of 12 (out of 12), 11 (out of 12), 11 (out of 12) and 11 (out of 12) females were confirmed with pregnancy (presence of implantations / presence of live or dead fetuses / evidence of parturition) with a fertility index of 100.0%, 91.7%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There were no statistically significant effects on female fertility indices of dosed groups when compared with vehicle control group.

Pre-coital Interval (Days): A total of 12 pairs were left for cohabitation initially from each tested dose group and vehicle control group. The mean pre-coital interval was 6.75, 8.08, 7.83 and 8.92 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in dosed groups when compared with the vehicle control group.

Gestation Length (Days): The mean gestation length [confirmation of mating to
parturition] was 22.58, 22.55, 22.55 and 22.73 days for groups G1, G2, G3 and G4,
respectively. There was no statistically significant difference in this parameter in any of the dosed groups when compared with the vehicle control group.

Gestation Index / Parturition Index (%): A total of 12 (out of 12), 11 (out of 11), 11
(out of 11), and 11 (out of 11) females were confirmed with live born pups with a
gestation / parturition index of 100%, 100%, 100% and 100% from group G1, G2, G3 and G4 respectively.

Pregnancy Index (%): A total of 12 (out of 12), 11 (out of 12), 11 (out of 12), and 11 (out of 12) females were confirmed with live born pups with a pregnancy index of 100.0%, 91.7%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in any of the dosed groups when compared with the vehicle control group.

Mean number of Implantations (No.): The mean number of implantations per litter was 11.75, 11.18, 10.91 and 11.55 from group G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in any of the dosed groups when compared with the vehicle control group.

Post implantation loss per litter (No.) and (%): The post implantation losses per litter was 0.50, 0.55, 0.09 and 0.45 with a percentage of 4.01, 4.33, 5.31 and 4.66 from group G1, G2, G3 and G4, respectively. There was no statistically significant
difference in this parameter in any of the dosed groups when compared with the
vehicle control group.

Postnatal losses on Lactation Day 13 (No.) and (%): There was no postnatal loss
noted in any of the litters from all the dosed and vehicle control groups.

Percentage of male/female offspring per litter (%): The percentage of male and female offspring per litter was 48.83 and 51.17 for G1, 55.51 and 44.49 for G2, 47.13 and 52.87 for G3 and 56.90 and 43.10 for G4 respectively. There was no statistically significant difference in this parameter in any of the tested dose groups when compared with the vehicle control group.

The data of 12 (out of 12 mated females), 11 (out of 12 mated females),
11 (out of 12 mated females) and 11 (out of 12 mated females) females confirmed with parturition from group G1, G2, G3 and G4, respectively, were considered for mean calculations and subjected to statistical analysis to analyse the maternal endpoints.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal behaviour was observed in any of the pups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth indexes were 99.40, 98.48, 100, and 100% in G1, G2, G3, and G4, respectively. Pup survival index was 100% in all groups on PND 4, 7, and 13.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant changes in serum thyroxine (T4) hormone levels were observed (examined in pups on PND 13).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No significant changes in AGD or AGD ratio (cube root body weight) were observed.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences of nipples in male pups examined on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes were observed in any of the adult animals or pups.

Histopathological findings:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

• TABLE 1. SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

                                                                                                                                                                                                                Refer Appendix 1

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^b: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	Day 1 to till termination: N (12)	0 (12)
G2, M & 87.5	12	Day 1 to till termination: N (12)	0 (12)
G3, M & 175	12	Day 1 to till termination: N (12)	0 (12)
G4, M & 350	12	Day 1 to till termination: N (12)	0 (12)

M: Male; N: Normal
a: observed daily once; b: observed twice daily

Table of Effects:

Group	Clinical Signs occurrence	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	No	NA	NA
G2	No	NA	NA
G3	No	NA	NA
G4	No	NA	NA

NA: Not Applicable

 

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

                         Refer Appendix 1

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^b No. of Mortalities (Total No. of Animals)
G1, F & 0	12	Day 1 to till termination: N (12)	0 (12)
G2, F & 87.5	12	Day 1 to till termination: N (12)	0 (12)
G3, F & 175	12	Day 1 to till termination: N (12)	0 (12)
G4, F & 350	12	Day 1 to till termination: N (12)	0 (12)

F: Female; N: Normal

a: observed daily once; b: observed twice daily

Table of Effects:

Group	Clinical Signs occurrence	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	No	NA	NA
G2	No	NA	NA
G3	No	NA	NA
G4	No	NA	NA

NA: Not Applicable

 

 

• SUMMARY OF BODY WEIGHT (g) RECORD

   Refer Appendix 2

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35
G1, M & 0	Mean	356.09	372.81	387.92	398.69	409.25	425.76
	±SD	32.00	32.70	30.15	27.25	26.40	27.23
	n	12	12	12	12	12	12
G2, M & 87.5	Mean	358.49	370.19	381.21	390.71	401.02	415.55
	±SD	32.01	34.46	34.35	34.44	37.75	42.38
	n	12	12	12	12	12	12
G3, M & 175	Mean	356.80	365.21	382.45	388.26	402.14	421.50
	±SD	32.59	34.49	34.76	33.04	30.90	42.93
	n	12	12	12	12	12	12
G4, M & 350	Mean	355.70	368.06	387.58	396.80	399.99	420.44
	±SD	30.80	32.95	32.47	31.78	32.65	32.27
	n	12	12	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

TABLE 2 (Contd…). SUMMARY OF BODY WEIGHT (g) RECORD

Refer Appendix 2

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21 #	28 #
G1, F & 0	Mean	251.78	264.14	280.34	281.47	301.49
	±SD	16.80	15.26	15.26	23.03	-
	n	12	12	12	5
G2, F & 87.5	Mean	253.35	262.96	278.62	281.35	281.79
	±SD	16.57	17.80	15.52	20.41	-
	n	12	12	12	6
G3, F & 175	Mean	253.98	265.26	285.68	291.18	-
	±SD	16.90	17.63	16.42	16.66	-
	n	12	12	12	6
G4, F & 350	Mean	253.12	263.60	281.77	282.04	298.38
	±SD	13.95	18.08	17.55	19.19	3.66
	n	12	12	12	7

F: Female; SD: Standard Deviation; n: Number of Animals; -: Not Applicable; #: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables.

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

• SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1

                                             Refer Appendix 3

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35
G1, M & 0	Mean	4.73	9.11	12.25	15.29	20.01
	±SD	1.73	3.82	5.04	6.20	7.67
	n	12	12	12	12	12
G2, M & 87.5	Mean	3.26	6.39	9.06	11.88	15.88
	±SD	2.91	3.73	3.84	3.96	4.81
	n	12	12	12	12	12
G3, M & 175	Mean	2.37	7.31	9.00	13.03	18.44
	±SD	2.51	5.03	5.49	7.01	10.52
	n	12	12	12	12	12
G4, M & 350	Mean	3.50	9.10	11.75	12.82	18.65
	±SD	3.18	5.10	5.52	8.60	9.35
	n	12	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

TABLE 3 (Contd…). SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1

  Refer Appendix 3

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21 #	1 to 28 #
G1, F & 0	Mean	5.00	11.49	12.75	17.34
	±SD	3.20	4.15	4.52	-
	n	12	12	5
G2, F & 87.5	Mean	3.79	10.07	9.30	11.64
	±SD	1.77	3.17	2.48	-
	n	12	12	6
G3, F & 175	Mean	4.47	12.57	13.15	-
	±SD	2.39	3.00	5.11	-
	n	12	12	6
G4, F & 350	Mean	4.08	11.29	11.41	12.52
	±SD	1.96	2.21	2.00	0.77
	n	12	12	7

F: Female; SD: Standard Deviation; n: Number of Animals; -: Not Applicable; #: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables.

 

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

• SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

  Refer Appendix 4

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
G1, M & 0	Mean	24.77	26.30
	±SD	2.43	1.77
	n	12	12
G2, M & 87.5	Mean	22.36	25.43
	±SD	2.59	3.00
	n	12	12
G3, M & 175	Mean	23.31	26.39
	±SD	2.10	1.38
	n	12	12
G4, M & 350	Mean	22.49	25.64
	±SD	1.80	1.00
	n	12	12

M: Male; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

TABLE 4 (Contd…). SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

  Refer Appendix 4

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
G1, F & 0	Mean	18.05	18.70
	±SD	2.08	1.83
	n	12	12
G2, F & 87.5	Mean	17.29	18.24
	±SD	1.48	2.06
	n	12	12
G3, F & 175	Mean	17.39	19.07
	±SD	1.50	0.86
	n	12	12
G4, F & 350	Mean	18.05	18.70
	±SD	2.08	1.83
	n	12	12

F: Female; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Increase/Decrease/No Effect	Significance	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA	NA
G2	No Effect	NA	NA	NA
G3	No Effect	NA	NA	NA
G4	No Effect	NA	NA	NA

NA: Not Applicable

 

• SUMMARY OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Refer Appendix 5

Determination of Oestrus Cyclicity during Pre-Mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		No. of Females with Complete Regular Oestrus Cycle	No. of Females with atleast one Irregular Oestrus Cycle	Average Length of Oestrus Cycle (Days)
G1, F & 0	12	n	12	0	Mean	4.44
		%	100.0	0.0	±SD	0.15
					n	12
G2, F & 87.5	12	n	12	0	Mean	4.46
		%	100.0	0.0	±SD	0.14
					n	12
G3, F & 175	12	n	12	0	Mean	4.43
		%	100.0	0.0	±SD	0.15
					n	12
G4, F & 350	12	n	12	0	Mean	4.44
		%	100.0	0.0	±SD	0.15
					n	12

F: Female; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Effect/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

TABLE 5 (Contd…). SUMMARY OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

          Refer Appendix 5

Determination of Oestrus Cyclicity during Mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		No. of Females with Complete Regular Oestrus Cycle	No. of Females with atleast one Irregular Oestrus Cycle	Average Length of Oestrus Cycle (Days)
G1, F & 0	12	n	6	0	Mean	4.44
		%	100.0	0.0	±SD	0.39
					n	6
G2, F & 87.5	12	n	7	0	Mean	4.52
		%	100.0	0.0	±SD	0.30
					n	7
G3, F & 175	12	n	8	0	Mean	4.69
		%	100.0	0.0	±SD	0.37
					n	8
G4, F & 350	12	n	7	0	Mean	4.55
		%	100.0	0.0	±SD	0.08
					n	7

 F: Female; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Effect/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF GESTATION BODY WEIGHT (g)

Refer Appendix 6

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	284.26	300.40	330.06	392.16
	±SD	18.62	18.48	21.44	21.73
	n	12	12	12	12
G2, F & 87.5	Mean	283.29	296.22	326.72	387.94
	±SD	16.64	17.28	19.13	21.02
	n	11	11	11	11
G3, F & 175	Mean	286.76	301.03	329.86	393.44
	±SD	21.06	23.00	22.63	22.36
	n	11	11	11	11
G4, F & 350	Mean	280.94	295.65	325.19	387.94
	±SD	17.62	18.83	20.56	23.36
	n	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams 

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) DURING GESTATION PERIOD

Refer Appendix 7

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	5.72	9.87	18.95
	±SD	1.92	2.05	4.51
	n	12	12	12
G2, F & 87.5	Mean	4.58	10.31	18.82
	±SD	1.59	1.98	4.00
	n	11	11	11
G3, F & 175	Mean	4.97	9.64	19.43
	±SD	1.88	1.55	4.46
	n	11	11	11
G4, F & 350	Mean	5.24	10.00	19.41
	±SD	1.45	1.64	4.88
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD

Refer Appendix 8

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	19.15	23.07	26.34
	±SD	0.78	1.84	2.22
	n	12	12	12
G2, F & 87.5	Mean	18.70	21.85	24.96
	±SD	1.23	1.61	1.66
	n	11	11	11
G3, F & 175	Mean	18.98	21.79	24.92
	±SD	0.61	1.05	1.63
	n	11	11	11
G4, F & 350	Mean	18.48	20.52*	23.25*
	±SD	0.99	1.86	2.59
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

*: Statistically significant (P<0.05) change than the concurrent vehicle control group

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	Decrease	No	NA

NA: Not Applicable

 

• SUMMARY OF LACTATION BODY WEIGHT (g)

Refer Appendix 9

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	13
G1, F & 0	Mean	309.42	314.59	327.96	339.36
	±SD	18.33	16.28	15.55	15.15
	n	12	12	12	12
G2, F & 87.5	Mean	305.41	310.08	321.42	336.29
	±SD	20.58	21.69	22.60	24.81
	n	11	11	11	11
G3, F & 175	Mean	305.06	311.55	321.66	333.13
	±SD	18.40	15.37	15.55	15.23
	n	11	11	11	11
G4, F & 350	Mean	301.50	309.75	319.89	332.04
	±SD	16.25	17.43	18.06	19.05
	n	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION PERIOD

Refer Appendix 10

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	1.72	4.29	3.49
	±SD	1.14	2.29	0.87
	n	12	12	12
G2, F & 87.5	Mean	1.52	3.66	4.61*
	±SD	1.01	1.12	1.19
	n	11	11	11
G3, F & 175	Mean	2.22	3.26	3.58
	±SD	2.74	1.17	0.63
	n	11	11	11
G4, F & 350	Mean	2.73	3.27	3.80
	±SD	1.02	0.79	1.43
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

*: Statistically significant (P<0.05) change than the concurrent vehicle control group

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	Increase	No	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

                                                                                                                

• SUMMARY OF FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD

Refer Appendix 11

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	29.25	32.24	36.60
	±SD	2.77	2.55	0.99
	n	12	12	12
G2, F & 87.5	Mean	29.69	33.07	35.77
	±SD	2.58	2.64	1.64
	n	11	11	11
G3, F & 175	Mean	29.93	33.21	36.30
	±SD	1.69	2.08	1.76
	n	11	11	11
G4, F & 350	Mean	28.78	33.55	36.27
	±SD	1.97	2.02	0.95
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF DELIVERY DATA (AT BIRTH) PER LITTER

Refer Appendix 12

Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)					Sex Ratio (M/F) at Birth	Live Birth Index (%)	% of offspring
			Male	Female	Total		Male	Female	Total		Undetermined	Male	Female	Total				Male	Female
G1, F & 0	Mean	11.33	5.50	5.75	11.25		0.08	0.00	0.08		0.00	0.00	0.00	0.00		1.05	99.40	48.83	51.17
	±SD	2.53	1.78	1.82	2.45		0.29	0.00	0.29		0.00	0.00	0.00	0.00		0.50	2.06	11.53	11.53
	n	12	12	12	12		12	12	12		12	12	12	12		12	12	12	12
G2, F & 87.5	Mean	10.82	5.91	4.73	10.64		0.00	0.18	0.18		0.00	0.00	0.00	0.00		1.50	98.48	55.51	44.49
	±SD	1.60	2.02	1.85	1.57		0.00	0.60	0.60		0.00	0.00	0.00	0.00		0.92	5.03	14.84	14.31
	n	11	11	11	11		11	11	11		11	11	11	11		11	11	11	11
G3, F & 175	Mean	10.82	5.09	5.73	10.82		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.95	100.00	47.13	52.87
	±SD	1.72	1.30	1.42	1.72		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.32	0.00	12.71	16.12
	n	11	11	11	11		11	11	11		11	11	11	11		11	11	11	11
G4, F & 350	Mean	11.09	6.36	4.73	11.09		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.66	100.00	56.90	43.10
	±SD	2.88	2.54	2.05	2.88		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.18	0.00	10.43	14.62
	n	11	11	11	11		11	11	11		11	11	11	11		11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams; M/F: Male/Female

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF LITTER OBSERVATION DURING LACTATION PERIOD

Refer Appendix 13

Group, Sex & Dose (mg/kg body weight/day)		Male Live Pups (No.)	Female Live Pups (No.)	No. of Live Pups At Birth		During Lactation Day 1 to 4											Sex Ratio (M/F) at LD 4	Pup Survival Index (%) LD 1 to 4
						Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
						Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	5.50	5.75	11.25		5.50	5.75	11.25		0.00	0.00	0.00		0.00	0.00	0.00	1.05	100.00
	±SD	1.78	1.82	2.45		1.78	1.82	2.45		0.00	0.00	0.00		0.00	0.00	0.00	0.50	0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12	12	12
G2, F & 87.5	Mean	5.91	4.73	10.64		5.91	4.73	10.64		0.00	0.00	0.00		0.00	0.00	0.00	1.50	100.00
	±SD	2.02	1.85	1.57		2.02	1.85	1.57		0.00	0.00	0.00		0.00	0.00	0.00	0.92	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11
G3, F & 175	Mean	5.09	5.73	10.82		5.09	5.73	10.82		0.00	0.00	0.00		0.00	0.00	0.00	0.95	100.00
	±SD	1.30	1.42	1.72		1.30	1.42	1.72		0.00	0.00	0.00		0.00	0.00	0.00	0.32	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11
G4, F & 350	Mean	6.36	4.73	11.09		6.36	4.73	11.09		0.00	0.00	0.00		0.00	0.00	0.00	1.66	100.00
	±SD	2.54	2.05	2.88		2.54	2.05	2.88		0.00	0.00	0.00		0.00	0.00	0.00	1.18	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams; M/F: Male/Female; LD: Lactation Day

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

TABLE 13 (Contd…). SUMMARY OF DELIVERY AND LITTER OBSERVATION DURING LACTATION PERIOD

Refer Appendix 13

Group & Dose (mg/kg body weight/day)	Animal No.	Mean no. of Live Pups (No.) on LD 4				Pups Sacrificed for Blood Collection on LD 4 (No.)				Live Pups (No.) on LD 4 after Sacrificed for Blood Collection				During Lactation Day 4 to 7												Sex Ratio (M/F) at LD 7	Pup Survival Index (%) during LD 4 to 7
														Live Pups per litter (No.)				Dead Pups per litter (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	5.50	5.75	11.25		0.00	1.08	1.08		5.50	4.67	10.17		5.50	4.67	10.17		0.00	0.00	0.00		0.00	0.00	0.00		1.41	100.00
	±SD	1.78	1.82	2.45		0.00	1.00	1.00		1.78	1.50	1.70		1.78	1.50	1.70		0.00	0.00	0.00		0.00	0.00	0.00		1.05	0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12		12	12	12		12	12	12		12	12
G2, F & 87.5	Mean	5.91	4.73	10.64		0.00	0.55	0.55		5.91	4.18	10.09		5.91	4.18	10.09		0.00	0.00	0.00		0.00	0.00	0.00		2.23	100.00
	±SD	2.02	1.85	1.57		0.00	0.82	0.82		2.02	1.72	0.83		2.02	1.72	0.83		0.00	0.00	0.00		0.00	0.00	0.00		2.96	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11
G3, F & 175	Mean	5.09	5.73	10.82		0.00	0.91	0.91		5.09	4.82	9.91		5.09	4.82	9.91		0.00	0.00	0.00		0.00	0.00	0.00		1.15	100.00
	±SD	1.30	1.42	1.72		0.00	1.04	1.04		1.30	1.33	0.94		1.30	1.33	0.94		0.00	0.00	0.00		0.00	0.00	0.00		0.42	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11
G4, F & 350	Mean	6.36	4.73	11.09		0.00	1.18	1.18		6.36	3.55	9.91		6.36	3.55	9.91		0.00	0.00	0.00		0.00	0.00	0.00		2.94	100.00
	±SD	2.54	2.05	2.88		0.00	0.98	0.98		2.54	1.75	1.97		2.54	1.75	1.97		0.00	0.00	0.00		0.00	0.00	0.00		3.12	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; M/F: Male/Female

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

 

TABLE 13 (Contd…). SUMMARY OF LITTER OBSERVATION DURING LACTATION PERIOD

Refer Appendix 13

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	Live Pups (No.) on LD 7				During Lactation Day 7 to 13											Sex Ratio (M/F) at LD 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	5.50	4.67	10.17		5.50	4.67	10.17		0.00	0.00	0.00		0.00	0.00	0.00	1.41	100.00
	±SD	1.78	1.50	1.70		1.78	1.50	1.70		0.00	0.00	0.00		0.00	0.00	0.00	1.05	0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12	12	12
G2, F & 87.5	Mean	5.91	4.18	10.09		5.91	4.18	10.09		0.00	0.00	0.00		0.00	0.00	0.00	2.23	100.00
	±SD	2.02	1.72	0.83		2.02	1.72	0.83		0.00	0.00	0.00		0.00	0.00	0.00	2.96	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11
G3, F & 175	Mean	5.09	4.82	9.91		5.09	4.82	9.91		0.00	0.00	0.00		0.00	0.00	0.00	1.15	100.00
	±SD	1.30	1.33	0.94		1.30	1.33	0.94		0.00	0.00	0.00		0.00	0.00	0.00	0.42	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11
G4, F & 350	Mean	6.36	3.55	9.91		6.36	3.55	9.91		0.00	0.00	0.00		0.00	0.00	0.00	2.94	100.00
	±SD	2.54	1.75	1.97		2.54	1.75	1.97		0.00	0.00	0.00		0.00	0.00	0.00	3.12	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; M/F: Male/Female

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF REPRODUCTIVE PERFORMANCE

Refer Appendix 14

Group, Sex & Dose (mg/kg body weight/day)	No. of Males with Evidence of Mating (Total No. of Males used for Mating)	Male Mating Index (%)		No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertiltiy Index (%)
G1, M & 0	11 (12)	91.7		11 (12)	91.7
G2, M & 87.5	11 (12)	91.7		11 (12)	91.7
G3, M & 175	12 (12)	100.0		12 (12)	100.0
G4, M & 350	10 (12)	83.3		10 (12)	83.3

M: Male

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE

Refer Appendix 14

Group, Sex & Dose                                     (mg/kg body weight/day)		Copulatory interval / Pre-coital interval (Mean Time to Mating) / Cohabitation Record					Gestation Length / Duration of Pregnancy (Days)
		Pre-coital Interval		Concieving Days (1 to 5)	Concieving Days (5 to More)
G1, F & 0	Mean	6.75	n	6	6		22.58
	±SD	5.33	%	50.0	50.0		0.51
	n	12					12
G2, F & 87.5	Mean	8.08	n	5	7		22.55
	±SD	5.12	%	41.7	58.3		0.52
	n	12					11
G3, F & 175	Mean	7.83	n	5	7		22.55
	±SD	5.04	%	41.7	58.3		0.52
	n	12					11
G4, F & 350	Mean	8.92	n	5	7		22.73
	±SD	5.73	%	41.7	58.3		0.47
	n	12					11

F: Female; SD: Standard Deviation; n: Number of Animals (Females confirmed with mating) / Dams confirmed with pregnancy

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE

Refer Appendix 14

Group, Sex & Dose (mg/kg body weight/day)	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)		No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertility Index (%)
G1, F & 0	12 (12)	100.0		12 (12)	100.0
G2, F & 87.5	12 (12)	100.0		11 (12)	91.7
G3, F & 175	12 (12)	100.0		11 (12)	91.7
G4, F & 350	12 (12)	100.0		11 (12)	91.7

F: Female

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

                                                                                                         

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE

Refer Appendix 14

Group, Sex & Dose (mg/kg body weight/day)	Pregnancy Index (%)				Gestation Index (%)				Parturition Index (%)
	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)		Females with Live Born Pups at Parturition	No. of Females with Evidence of Pregnancy	Gestation Index (%)		No. of females littered	No. of Females with Evidence of Pregnancy	Parturition Index (%)
G1, F & 0	12	12	100.0		12	12	100.0		12	12	100.0
G2, F & 87.5	11	12	91.7		11	11	100.0		11	11	100.0
G3, F & 175	11	12	91.7		11	11	100.0		11	11	100.0
G4, F & 350	11	12	91.7		11	11	100.0		11	11	100.0

F: Female

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE

Refer Appendix 14

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)					Post-natal Loss (%)
		No. of Implantations	No. of Viable Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)		Total No. of Deaths/ Cannibalized during Lactation Period	Post-natal Loss (%)
G1, F & 0	Mean	11.75	11.25	0.50	4.01		0.00	0.00
	±SD	2.53	2.45	0.80	6.33		0.00	0.00
	n	12	12	12	12		12	12
G2, F & 87.5	Mean	11.18	10.64	0.55	4.51		0.00	0.00
	±SD	1.60	1.57	1.04	8.42		0.00	0.00
	n	11	11	11	11		11	11
G3, F & 175	Mean	10.91	10.82	0.09	0.91		0.00	0.00
	±SD	1.64	1.72	0.30	3.02		0.00	0.00
	n	11	11	11	11		11	11
G4, F & 350	Mean	11.55	11.09	0.45	4.12		0.00	0.00
	±SD	2.70	2.88	0.82	7.71		0.00	0.00
	n	11	11	11	11		11	11

F: Female; n: Number of Dams

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

           Refer Appendix 15

Group, Sex & Dose (mg/kg body weight/day)		Testes	Epididymes	Prostate + Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid #
G1, M & 0	Mean	3.5615	1.4352	3.3812	0.0230
	±SD	0.2510	0.0970	0.4057	0.0029
	n	12	12	12	12
G2, M & 87.5	Mean	3.6884	1.4706	3.2724	0.0210
	±SD	0.1675	0.1051	0.3946	0.0018
	n	12	12	12	12
G3, M & 175	Mean	3.4426	1.4507	3.3433	0.0250
	±SD	0.3112	0.0971	0.4443	0.0037
	n	12	12	12	12
G4, M & 350	Mean	3.6953	1.4694	3.4254	0.0224
	±SD	0.2992	0.1037	0.3306	0.0029
	n	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation

Table of Effects:

Organ Weight	Sex	Dose	Increase/Decrease/ No Effect	Significance in terms of relative organ Weight Increase/Decrease/ No Effect	Correlation with Gross Necropsy/Histopathology Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
Testes	M	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No effect	NA	NA
Epididymis	M	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No effect	NA	NA
PSC	M	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No effect	NA	NA
Thyroid along with parathyroid	M	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No effect	NA	NA

NA: Not Applicable

 

TABLE 15 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

 Refer Appendix 15

Group, Sex & Dose (mg/kg body weight/day)		Ovaries	Thyroid along with parathyroid #
G1, F & 0	Mean	0.1264	0.0201
	±SD	0.0323	0.0033
	n	12	12
G2, F & 87.5	Mean	0.1181	0.0214
	±SD	0.0192	0.0028
	n^@	11	11
G3, F & 175	Mean	0.1355	0.0205
	±SD	0.0317	0.0035
	n^@	11	11
G4, F & 350	Mean	0.1063	0.0209
	±SD	0.0256	0.0030
	n^@	11	11

F: Female; SD: Standard Deviation; n: Number of Dams; #: Weighed post fixation; @: the data of non-pregnant female from G2, G3 and G4 each was excluded from mean calculations and statistical analysis

Table of Effects:

Organ Weight	Sex	Dose	Increase/Decrease/No Effect	Significance in terms of relative organ Weight Increase/ Decrease/ No Effect	Correlation with GrossNecropsy/ Histopathology Increase/ Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
Ovaries	F	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No Effect	NA	NA
Thyroid along with parathyroid	F	87.5 mg/kg, 175 mg/kg, 350 mg/kg	No Effect	No Effect	No Effect	NA	NA

NA: Not Applicable

 

 

• SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
  TERMINAL BODY WEIGHT (%) RECORD

                                                                                                                                                                                                              Refer Appendix 16

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Testes	Epididymes	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid
G1, M & 0	Mean	409.46	0.8717	0.3511	0.8249	0.0057
	±SD	25.71	0.0659	0.0225	0.0729	0.0009
	n	12	12	12	12	12
G2, M & 87.5	Mean	398.21	0.9345	0.3726	0.8288	0.0053
	±SD	39.84	0.0980	0.0450	0.1264	0.0006
	n	12	12	12	12	12
G3, M & 175	Mean	399.68	0.8687	0.3664	0.8426	0.0063
	±SD	44.44	0.1070	0.0403	0.1228	0.0010
	n	12	12	12	12	12
G4, M & 350	Mean	402.31	0.9225	0.3663	0.8528	0.0056
	±SD	28.75	0.0950	0.0294	0.0755	0.0008
	n	12	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Organ Weight	Sex	Dose	Increase/Decrease/No Effect	Significance in terms of relative organ Weight Increase/Decrease/No Effect	Correlation with Gross Necropsy/Histopathology Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
Testes	M	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA
Epididymis	M	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA
PSC	M	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA
Thyroid along with parathyroid	M	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA

NA: Not Applicable

 

TABLE 16 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Refer Appendix 16

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Ovaries	Thyroid along with parathyroid
G1, F & 0	Mean	329.12	0.0383	0.0061
	±SD	16.13	0.0089	0.0009
	n	12	12	12
G2, F & 87.5	Mean	324.79	0.0364	0.0066
	±SD	26.62	0.0048	0.0010
	n	11	11	11
G3, F & 175	Mean	323.13	0.0420	0.0063
	±SD	17.03	0.0101	0.0010
	n	11	11	11
G4, F & 350	Mean	322.15	0.0329	0.0065
	±SD	20.46	0.0072	0.0009
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams

Table of Effects:

Organ Weight	Sex	Dose	Increase/ Decrease/ No Effect	Significance in terms of relative organ Weight Increase/ Decrease/ No Effect	Correlation with Gross Necropsy/ Histopathology Increase/ Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
Ovaries	F	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA
Thyroid along with parathyroid	F	87.5 mg/kg, 175 mg/kg  350 mg/kg	No Effect	No Effect	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD -

Refer Appendix 17

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	54.775
	±SD	5.262
	n	12
G2, M & 87.5	Mean	52.626
	±SD	4.612
	n	12
G3, M & 175	Mean	56.175
	±SD	7.082
	n	12
G4, M & 350	Mean	53.380
	±SD	3.874
	n	12

 M: Male; SD: Standard Deviation; n: Number of Animals

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF PUP OBSERVATIONS DURING POST-NATAL PERIOD

Refer Appendix 18

Group, Sex & Dose                   (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.)*	PND 4 to 7^$	PND 7 to 13^$
G1, F & 0	No. of Dams / Litters^#	12	12	7	12	12
	No. of Live Pups	135	135	13	122	122
	Pup Observation/ No. of Pups observed	N/135	N/135	-	N/122	N/122
G2, F & 87.5	No. of Dams / Litters^#	11	11	4	11	11
	No. of Live Pups	117	117	6	111	111
	Pup Observation/ No. of Pups observed	N/117	N/117	-	N/111	N/111
G3, F & 175	No. of Dams / Litters^#	11	11	5	11	11
	No. of Live Pups	119	119	10	109	109
	Pup Observation/ No. of Pups observed	N/119	N/119	-	N/109	N/109
G4, F & 350	No. of Dams / Litters^#	11	11	7	11	11
	No. of Live Pups	122	122	13	109	109
	Pup Observation/ No. of Pups observed	N/122	N/122	-	N/109	N/109

F: Female; N: Normal; PND: Post-natal Day; #: confirmed with live pups

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table of Effects:

Group	Effect/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF MEAN PUP WEIGHT (g) PER LITTER DURING LACTATION PERIOD

Refer Appendix 19

Group, Sex & Dose                   (mg/kg body weight/day)		PND 1			PND 4			PND 7			PND 13
		Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)
		Male	Female		Male	Female		Male	Female		Male	Female
G1, F & 0	Mean	6.34	5.49		10.73	9.82		15.15	13.86		26.37	24.61
	±SD	0.49	0.32		0.29	0.30		0.79	1.14		0.53	0.23
	n	12	12		12	12		12	12		12	12
G2, F & 87.5	Mean	5.99	5.29		10.85	9.82		15.44	14.01*		26.32	24.69
	±SD	0.33	0.18		0.41	0.43		0.56	0.67		0.37	0.16
	n	11	11		11	11		11	11		11	11
G3, F & 175	Mean	6.19	5.41		10.85	9.93		15.47	14.17*		26.39	24.87
	±SD	0.42	0.21		0.35	0.45		0.71	0.86		0.45	0.46
	n	11	11		11	11		11	11		11	11
G4, F & 350	Mean	6.10	5.41		10.83	10.00		15.51	14.20*		26.22	24.92
	±SD	0.28	0.16		0.55	0.56		0.65	0.99		0.45	0.52
	n	11	11		11	11		11	11		11	11

F: Female; SD: Standard Deviation; n: Number of Litters; PND: Post-Natal Day

*: Statistically significant (P<0.05) change than the concurrent vehicle control group

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	Decrease	No	NA
G3	Decrease	No	NA
G4	Decrease	No	NA

NA: Not Applicable

 

• SUMMARY OF MEAN PUP ANO-GENITAL DISTANCE (AGD) MEASUREMENT (mm) AND
  ANO-GENITAL DISTANCE (AGD) RATIO PER LITTER ON POST-NATAL DAY 4

Refer Appendix 20

Group, Sex & Dose (mg/kg body weight/day)		Mean Male Pup AGD Measurement (mm)	Mean Female Pup AGD Measurement (mm)		Mean Male Pup AGD Ratio	Mean Female Pup AGD Ratio
G1, F & 0	Mean	4.38	2.40		1.99	1.12
	±SD	0.13	0.11		0.06	0.06
	n	12	12		12	12
G2, F & 87.5	Mean	4.32	2.43		1.95	1.13
	±SD	0.08	0.15		0.05	0.07
	n	11	11		11	11
G3, F & 175	Mean	4.36	2.47		1.97	1.15
	±SD	0.09	0.07		0.05	0.04
	n	11	11		11	11
G4, F & 350	Mean	4.35	2.39		1.97	1.11
	±SD	0.07	0.11		0.06	0.06
	n	11	11		11	11

F: Female; SD: Standard Deviation; n: Number of Litters; AGD: Ano-genital Distance

Table of Effects:

Group	Increase/Decrease/No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

 

 

 

• SUMMARY OF MALE PUP NIPPLE/AREOLAE RETENTION (no.) RECORD PER LITTER

Refer Appendix 21

Group & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Post-natal Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	12
G2 & 87.5	Mean	0.00
	±SD	0.00
	n	11
G3 & 175	Mean	0.00
	±SD	0.00
	n	11
G4 & 350	Mean	0.00
	±SD	0.00
	n	11

SD: Standard Deviation; n: Number of Litters

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

• SUMMARY OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - POST-NATAL DAY 13 PUPS (PND)

                                                                                                             Refer Appendix 22

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	49.644
	±SD	5.616
	n	12
G2 & 87.5	Mean	49.473
	±SD	3.935
	n	11
G3 & 175	Mean	47.117
	±SD	3.680
	n	11
G4 & 350	Mean	46.835
	±SD	4.650
	n	11

SD: Standard Deviation; n: Number of Litters; PND: Post-Natal Day

Table of Effects:

Group	Increase/Decrease/ No Effect	Dose Related (Yes/No)	Vehicle Related (Yes/No)
G1	NA	NA	NA
G2	No Effect	NA	NA
G3	No Effect	NA	NA
G4	No Effect	NA	NA

NA: Not Applicable

 

Applicant's summary and conclusion

Conclusions:

The study-derived NOAEL for general, reproductive, and developmental toxicity was 350 mg/kg bw/day.

Executive summary:

A reproduction/developmental toxicity screening test as per the GLP compliance and in accordance with OECD TG 421 was performed using male and female Sprague Dawley rats for assessing the potential of the test chemical to cause adverse effect on the reproductive potential and effects on the developing fetuses. In this study 12 animals per sex per group were included which were grouped into 4 groups i.e. 0 mg/kg bw (Vehicle Control Group), 87.5 mg/kg bw (Low Dose Group), 175 mg/kg bw (Mid Dose Group) and 350 mg/kg bw (High Dose Group), respectively. Males were dosed for a period of 35 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period, while females were dosed for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 49 to day 67 ). The vehicle and test chemical formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. All the animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The weekly body weight and weekly feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. All the males were evaluated for reproductive performance such as, mating and fertility index. All the dams were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All the females were evaluated for oestrus cyclicity during premating, mating treatment periods and at termination. The body weight and feed consumption was recorded for all the females during gestation day 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. The terminal body weight for all the animals was measured on the day of the termination (LD 14). At birth/litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all the litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated. The pups were observed once daily for external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method. There were no clinical signs of toxicity and no mortality/morbidity noted at all in any of the tested dose groups of either sex animals. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all thein any of the tested dose groups of both the either sex. There were no changes noted in mean serum thyroxine hormone (T4) levels at all in any of the tested dose group of males. The absolute and relative organ weights did not reveal any changes at all thein any of the tested dose groups. No macroscopic changes noted during conduct of necropsy in at all the vehicle control and tested dosed groups of both either the sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test chemical related microscopic changes. For reproduction toxicity end points, there were no effects noted in male mating and fertility indexes of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length in at all tested all dosed groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption were observed all dosed groups during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by the test chemical at all the tested in all treated dose groups. For developmental toxicity end points, there were no external anomalies noted and no test chemical-related mortalities among pups noted were observed in pups during post-natal period at doses tested. at all the tested dose group litters. There were no test chemical-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups at doses tested. There were no occurrences of nipples in male pups of vehicle control and treated groups on PND 13 examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters at any doses tested. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose groups up litters. Thus, based on all the available data, it was concluded that the test chemical did not induce any systemic or reproductive toxicity in all animals in the tested dose groups and therefore the NOAEL for the test chemical was considered to be 350 mg/kg bw/day viz. the highest dose tested for the study.